Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

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Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

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Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

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Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Abstract

LOVTRAP is an optogenetic approach for reversible light-induced protein dissociation using protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in the dissociation constant (Kd). By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins' access to the cell edge, demonstrating a naturally occurring 3-mHz cell-edge oscillation driven by interactions of Vav2, Rac1, and PI3K proteins.

(a) LOV2 is anchored to the mitochondrial outer membrane. The protein of interest (POI) is attached to Zdk, a reagent that binds selectively to the dark state of LOV2. The POI is sequestered at mitochondria in the dark and released when cells are irradiated. (b) Thirteen residues along the first and second helices of the Z domain from protein A were randomized to generate a variable surface (left). Two consensus sequences bound specifically to the dark state of LOV2 (right). (c) Binding of Zdk isoforms to LOV2’s dark state (C450A mutant) versus lit state (I539E mutant) (n=4 for each curve). Dark versus lit affinities of Zdk1 (black), Zdk2 (red) and Zdk3 (blue) were 26.2±2.2 nM/>4 μM, 17.0±3.9 nM/761±78 nM, and 11.4±3.6 nM/537±37 nM, respectively. Error bars are the s.d. of replicates. (d) The amount of protein released upon irradiation depends upon the relative expression levels of the mitochondrial anchor and the POI. This model shows the effects of using Zdk1 on the POI and LOV2 at the mitochondria (red), or vice-versa (blue). Optimal response is seen when the protein attached to the mitochondria is in 4–10 fold excess over the POI. LOV2 on the mitochondria produces less free POI in the dark, and Zdk on mitochondria leads to more complete release. (e) Crystal structure of the LOV2-Zdk1 complex. Zdk1 (magenta) docks on the core domain of LOV2 (green) and the C-terminal residues of the LOV2 Jα helix (blue).